The goal of this project is to increase our understanding of prion propagation, which will help us to understand prion pathology and find treatments for these diseases. Prion diseases encompass a range of genetic, spontaneous, and transmitted neuropathologies that affect humans and a number of commercially important animals. At present, there is no known treatment or cure for prion diseases, in part because the mechanisms underlying prion pathology and the processes required for prion propagation are not well understood. Our understanding of prion pathogenesis would be greatly enhanced by identifying the auxiliary factors that are necessary for prion propagation. Numerous lines of evidence (including work in transgenic mice, cell culture infection assays, analogous factors in yeast prion propagation, and in vitro conversion assays) suggest that additional factors besides the primary sequence and expression level of the prion protein influence prion propagation. However, despite years of study, our understanding of this process in mammals is still limited. In part, this lack of progress may be due to the selection of candidates based on individual, low throughput studies. In aim one, we will harness the power of RNA interference to identify potential factors that modulate prion propagation in high throughput cell culture assays, and confirm our most promising hits in knockout mice. Successfully identifying mammalian prion propagation factors would not only provide new drug targets, but may allow us to explain why some cell lines are recalcitrant to prion infection despite high levels of prion protein expression In aim two we will use RNAi on a panel of cell lines infected with several prion strains to identif which of these potential prion propagation factors are always necessary for prion propagation and which of these factors are only necessary for specific strains. In aim three we will validate a subset of these potential prion propagation factors using existing mouse knockout lines. Increasing evidence that other neurodegenerative diseases are caused by a prion-like mechanism raises the possibility that these results could have a broad impact on human health.